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Obesity Research Center (G.C.Y., B.E.C.), Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118; and Immunology Division (H.M.K.), Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Gordon C. Yaney, Ph.D., Boston Medical Center, Obesity Research Center, 88 E. Newton Street, Boston, Massachusetts 02118. E-mail: gyaney{at}acs.bu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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found in the particulate
fraction. These data are consistent with increased cytosolic long-chain
acylCoA-activating aPKC isoforms resulting in stimulation and/or
potentiation of glucose-induced insulin secretion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A potentially important consequence of this malonyl CoA mediated
metabolic switch from FFA to glucose oxidation is the rapid synthesis
of complex lipids such as phosphatidic acid (PA) (9, 10, 11) or
diacylglycerol (DAG) (9, 10, 11, 12), and triglycerides (TG) (10, 13) and
is unlikely to occur without an increase in cytosolic LC-CoA levels.
Glucose metabolism, via the combination of 
-glycerophosphate and
cytosolic LC-CoA, leads to the de novo formation of PA and
DAG (10, 12), which are known to activate protein kinase C (PKC)
in vitro (14, 15).
PKC is now known to be a family of enzymes that can be divided into three classes based on their cofactor requirements (14, 16). All classes of the enzyme require the acidic phospholipid, phosphatidylserine (PS), for their activation. The conventional class (cPKC) requires PS, DAG, and Ca2+ for optimal activity, the novel class (nPKC) does not require Ca2+, and the atypical class (aPKC) requires only PS. Multiple PKC isoforms, representative of the three known classes, are expressed in the pancreatic ß-cell (17). Selective down-regulation of certain isoforms by overnight exposure to the activator phorbol myristate acetate (PMA) has also been demonstrated without inhibiting glucose-stimulated secretion (17). The aPKC isoforms are not stimulated or down-regulated by phorbol esters, and because the membrane concentration of PS is unchanged during cell activation the physiologic regulation of aPKC isoforms is uncertain.
We report here that PKC activity in ß-cell extracts was regulated by LC-CoA derivatives. Support was obtained for a model of nutrient-stimulated insulin secretion in which increases in cytosolic LC-CoA levels would stimulate aPKC and cPKC activity and inhibit nPKC activity as part of stimulus-secretion coupling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Secretion and insulin assays
HIT-T15 cells were grown in 24-well plates and used 2–4
days after passaging for secretion assays. Cells were washed twice in
Krebs-Ringer bicarbonate containing 2 mM
CaCl2 and 0.05% BSA and buffer with 10
mM HEPES at pH 7.4. The cells were preincubated with the
above KRB for 30 min at 37 C. The buffer was removed and the cells were
incubated for 30 min under the conditions cited in the figure legends.
Down-regulation of PKC activity was accomplished by overnight exposure
to 500 nM phorbol myristate acetate (PMA) containing 0.1%
final concentration of dimethylsulfoxide (DMSO) as vehicle. Control
plates were exposed to DMSO alone. Cooling the plate on ice was used to
stop further secretion. An aliquot was removed, spun to remove any
cells, and assayed for determination of insulin content. Insulin was
assayed by RIA using the double antibody assay protocol for rat insulin
distributed by Linco Research, Inc. (St. Charles,
MO).
Complexing of fatty acids to BSA
Oleate was complexed to essentially fatty acid-free BSA from
Sigma (St. Louis, MO) for use in secretion studies as
previously described (2). Final concentration of BSA was 14.5
µM complexed with 100 µM oleate resulting
in approximately 7 molecules of fatty acid per molecule of BSA.
Palmitate was used as a prepared complex of 5 molecules of fatty acid
per molecule of BSA as obtained from Sigma resulting in a
concentration for palmitate of approximately 74 µM with
the BSA used at a final concentration of 14.5 µM.
Protein kinase C assay
Cell extracts were prepared by adding 250 µl of extraction
buffer (50 mM Tris, pH containing 50 mM EGTA,
20 mM phenylmethylsulfonyl fluoride, 2 mg/ml of leupeptin
and aprotinin) to a T-25 flask containing approximately 3 x
106 cells. The cells were collected and sonicated
(3 x 20 sec) on ice using a microtip probe with a Branson Cell
Disrupter at 20% power level. The ruptured cells were spun at 700
x g to remove cell debris and the supernatant either used
to measure PKC activity or centrifuged at 110,000 x g
for 60 min. The use of homogenates rather than purified enzymes, has
the advantage of containing the relevant isoforms and modifiers, if
any.
Brain PS and 1,2-dioleoyl-sn-glycerol (DAG) stocks were dissolved in chloroform. Either PS alone or PS and DAG were combined and evaporated under N2 in glass tubes. Five hundred microliters of water was added and the tube was vortexed to hydrate the lipid layer then sonicated for 30 sec. This was done at room temperature using a microtip probe at a 10% power level with a Branson Cell Disrupter. The lipid micelles were held at 30 C until used. Phosphatidic acid (PA) and LC-CoA esters were made up as 10x stocks in water.
The assay used was based on the method of Majumdar et al. (19) with the incubation done in 96-well plates at a final volume of 50 µl. The reaction was run at 30 C for 15 min. Kinase activity was found to be linear up to 30 min, and proportional to the amount of extract added. The reaction was stopped by cooling on ice and adding 10 µl of 75 mM H3P04 to each well. Following a 30-min incubation, 20 µl from each was spotted on a 2 cm x 2 cm square of Whatman P81 paper and washed twice in distilled water containing 75 mM H3P04. The paper was air-dried and Cerenkov counting was used to determine 32P-phosphorus incorporation.
Each reaction had 5 µl of the following: reaction buffer
(0.25 M Tris, 0.1 M
MgCl2, 0.1% Triton-X100, pH 7.5), histone IIIs
(1.6 mg/ml in water), PKC extract (0.25 µg/µl), lipid sonicates (PS
at 200 µg/ml or PS/DG at 200/10 µg/ml), EGTA (4 mM) and
32P- ATP (500 µM) containing 1
µCi of tracer. The final volume of 15 µl was made up with
CaCl2 (6 mM) or LC-CoA stocks and
water. Ca2+-selective electrode and
Ca2+/EGTA standards were used to determine that
without the addition of CaCl2 the
Ca2+ was less than 10 nM in the
reaction mixture. The addition of 6 mM
CaCl2 increased free calcium to 10
µM. The protein content of the cell extract averaged 6
µg/µl and was diluted 250-fold in the assay.
Designation of kinase activity to PKC class
Activity was attributed to each class of PKC based on cofactor
requirements. The conventional class (cPKC) requires PS, DAG and
Ca2+ for optimal activity, the novel class (nPKC)
does not require Ca2+, and the atypical class
(aPKC) requires only an acidic phospholipid such as PS. Background
activity was defined as the phosphorylation seen in the presence of
EGTA or EGTA plus CaCl2. The EGTA background was
subtracted from conditions for aPKC or nPKC and the
EGTA/CaCl2 background was subtracted from
conditions for cPKC. aPKC was defined as the incremental activity over
background seen with PS. nPKC activity was defined as the incremental
activity over aPKC seen with DAG, while cPKC was defined as the
incremental effect over nPKC caused by Ca2+. The
effects of the LC-CoA esters were expressed as % of these
activities.
The various conditions were designated as follows: no Ca2+ background = 1, high Ca2+ background = 2, no Ca2+ + PS = 3, no Ca2+ + PS + DAG = 4, no Ca2+ + PS + LC-CoA = 5, no Ca2+ + PS + DAG/LC-CoA = 6, high Ca2+ + PS + DAG = 7, high Ca2+ + PS + DAG/LC-CoA = 8
Therefore, PKC classes were defined as follows: aPKC = 3–1, and the effect of LC-CoA = 6–1 nPKC = 4–3, and the effect of LC-CoA = 6–5 cPKC = () - (), and the effect of LC-CoA = () - ()
Western blotting
HIT cell extracts were prepared by adding 250 µl of the PKC
assay extraction buffer to a T-25 flask containing approximately 3
x 106 cells. The cells were sonicated (3 x
20 sec) on ice using a microtip probe with a Branson Cell Disrupter at
20% power level and spun at 700 x g to remove
unbroken cells. The remaining mixture was centrifuged at 110,000
x g for 60 min and the supernatant was designated high
speed supernatant. The resulting pellet was extracted in an equal
volume of extraction buffer containing 0.25% (vol/vol) of Triton-X
100. Following a second 110,000 x g spin the resulting
fractions were designated soluble and insoluble pellets with the
insoluble pellet resuspended by sonication in 250 µl of extraction
buffer. Transfer of protein to nitrocellulose paper was done
electrophoretically using a semidry transfer apparatus from Owl
Scientific (Cambridge, MA). Transfer buffer of Tris-HCl, pH 7.4
containing SDS and 20% methanol was used and the transfer performed
with constant current of 200 mA for 2 h at room temperature. Blots
were probed with isozyme specific polyclonal antibodies. With the
exception of anti-PKC-
, which was from Transduction Laboratories, Inc. (Lexington, KY), the antibodies were
purchased from Santa Cruz Biochemicals (Santa Cruz, CA)
and used as described by them. The secondary antibody was goat
antirabbit IgG conjugated to horseradish peroxidase purchased from
Roche Molecular Biochemicals (Indianapolis, IN) and used
at a dilution of 1:5000. The specificity of the interaction was
assessed by using the isoform specific blocking peptide provided.
Visualization of the secondary antibody was achieved using the enhanced
chemiluminescence (ECL) kit of Amersham Pharmacia Biotech
(Buckinghamshire, UK).
Intracellular translocation of PKC-
HIT cells were preincubated in KRB buffer without glucose at 37
C for 45 min and then incubated with KRB containing no glucose (basal),
5 mM glucose (glucose) or 5 mM glucose and a
7:1 complex (mol/mol) of oleate and fatty acid free BSA (glucose +
oleate). The cells were then quick-frozen in liquid nitrogen at 1, 3,
or 5 min after the start of incubation. Cell fractions of cytosol and
particulate (membranes) were prepared following sonication in the
previously mentioned extraction buffer and centrifuged at 160,000
x g for 60 min. Pellets were resuspended by sonication in
extraction buffer containing 0.1% TX-100. Protein content was
determined using the detergent-compatible protein assay of
Bio-Rad Laboratories, Inc. (Richmond, CA). Proteins
from soluble and particulate fractions were separated on SDS-gels,
transferred to nitrocellulose, probed with a PKC- isozyme-specific
antibody and visualized as outlined above. Films were digitized using
an Agfa flat-bed scanner and Adobe Photoshop, while the density of the
bands was quantitated using NIH Image, release 5.0.
Statistics
One-way ANOVA was performed followed by Student’s-Newman-Keuls
multiple comparisons tests for the analysis of concentration dependent
effects of LC-CoA. Unpaired two-tailed Student’s t tests
were performed for the comparison of a single concentration of LC-CoA
to its control value. A "P" value equal to or less than 0.05 was
considered significant. The program used was InStat 2.0 for the
Macintosh.
Materials
Phosphatidylserine, dioleoyl 1,2-diacylglycerol and phosphatidic
acid were obtained from Avanti Polar Lipids, Alabaster AL. Long chain
fatty acyl CoA esters were from either Amersham Pharmacia Biotech or Sigma. Histone IIIS, fatty acid
free BSA, oleate and BSA/palmitate complex (5:1 mol/mol), selenious
acid, glutathione, Dulbecco’s PBS, and penicillin/streptomyocin
mixture were from Sigma. Anion exchange paper, P81, was
from Whatman. 32P-ATP at 6000
Ci/mmol was obtained biweekly from NEN Life Science Products (Boston, MA). Culture medium RPMI-1640 and trypsin/EDTA
were from Life Technologies, Inc. (Gaithersburg, MD). FBS
was from HyClone Laboratories, Inc. (Salt Lake City, UT).
The Ca2+-selective electrode from ORION (Boston,
MA), used with Ca2+ and EGTA standards were from
World Precision Instruments (Sarasota, FL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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demonstrated
Ca2+-independent nPKC activity (open
bars) in the presence of PS and DAG. The level of nPKC activity
decreased when the concentration of DAG present was decreased. The
addition of 10 µM Ca2+
resulted in a significant increase in activity (Fig. 1, shaded
bar) at both concentrations of DAG (P < 0.001),
attributable, by definition, to cPKC. This value of free
Ca2+, while higher than the commonly reported
value occurring in the cytosol of activated cells, is within the range
of concentrations predicted to occur in the "active zones" of the
ß-cell plasma membrane surrounding the mouth of L-type calcium
channels (20).
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, PMA-down-regulated). Second, a large part of
the Ca2+-independent PKC activity (nPKC) was
lost. However, the nPKC activity that remained was also stimulated by
increasing DAG. Third, down-regulation did not inhibit, but rather
enhanced, the fold change in insulin secretion due to glucose
stimulation compared with control cells as seen in Fig. 5 (5.8 ±
0.2 vs. 4.2 ± 0.2, P < 0.002),
implying that cPKC may not be required for nutrient-stimulated
secretion.
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, Western blots demonstrated the
presence of aPKC isoforms and 
. Second, the addition of PS
caused a small, but significant increase in kinase activity above
background (6% ± 2, n = 24, P < 0.01).
Background activity is defined as the kinase activity seen without any
lipid additions to the assay. Third, the addition of phosphatidic acid
(PA) strongly potentiated the activity seen with PS as illustrated by a
580% ± 170 (n = 4) increase above that found with PS alone. PA
alone gave a 24% ± 10 (n = 6) increase above background
activity. Fourth, the potentiation of PS by PA was unchanged by
PMA-induced down-regulation (630% ± 150, n = 3). Therefore, the
stimulation of kinase activity observed in the absence of
Ca2+ and the lack of effect of PKC
down-regulation are consistent with the activation of aPKC isoforms by
PA reported in homogenates of COS cells in which the aPKC isoform,
PKC-, had been heterologously overexpressed (15).
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show the effects of palmitoyl-CoA on activity of the three classes of
PKC isoforms. Palmitoyl-CoA stimulated aPKC in a
concentration-dependent manner resulting in a 5-fold increase in
activity over PS alone with a half maximal effect at approximately 3
µM (Fig. 2A). In contrast, only 3 µM
palmitoyl-CoA resulted in a small increase in nPKC activity (Fig. 2B),
while higher concentrations were without effect. Lastly, this LC-CoA
ester resulted in a modest concentration-dependent stimulation of cPKC
activity with no significant differences between 10 µM
and 30 µM (Fig. 2C). Note the different scale used in
panel A vs. panels B and C. These data suggest that the
predominate effect of palmitoyl-CoA was to stimulate aPKC activity.
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. The short-chain length esters,
malonyl-CoA and succinyl-CoA, had no effect on aPKC (panel A) or a
slight stimulation of nPKC activity (panel B). Like palmitoyl-CoA, 10
µM myristoyl-CoA stimulated aPKC activity approximately
4-fold. The longer chain length monounsaturated LC-CoA, oleoyl-CoA
increased this activity approximately 8-fold. A similar stimulation
(750% ± 80, n = 5) was seen in extracts from PKC-down regulated
ß-cells, using 10 µM oleoyl-CoA. In contrast to
palmitoyl-CoA, which had little effect on nPKC activity, both 10
µM myristoyl-CoA and oleoyl-CoA inhibited this activity
by approximately 50% (Fig. 3B). Oleoyl-CoA caused a similar inhibition
of nPKC activity after PKC-down-regulation by PMA (51% ± 6, n =
4). These data confirm the ability of common LC-CoA esters to stimulate
aPKC and highlight different effects (no effect vs.
inhibition) on novel PKC isoforms.
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). nPKC activity varied over a wide
range depending on the DAG concentration used in the assay as
illustrated in Fig. 1. The nPKC activity was three times as great for
the assays containing 0.66 mol % DAG compared with the lower
concentration used. However, the half-maximal value of inhibition due
to oleoyl-CoA was not significantly altered by variations in the mol %
DAG used in the assay as evidenced by an IC50 of
9 or 12 µM for 0.22 and 0.66 mol %, respectively (Fig. 4A). These IC50 values were not significantly
different. Examination of the interaction between oleoyl-CoA and DAG
was extended by holding oleoyl-CoA at 10 µM and
increasing the mol % DAG in the mixed micelles over a wider range
(Fig. 4B). Inhibition of nPKC activity by oleoyl-CoA was independent of
the mol % DAG used over a 30-fold range.
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The effect of oleate and palmitate on glucose-stimulated insulin
secretion was evaluated to find out whether the two fatty acids had
different effects on secretion, as their LC-CoA esters had on nPKC
activity, or similar effects on secretion as the esters had on aPKC
activity. These FFA are converted to their LC-CoA esters in the
ß-cell (2). The FFA was delivered to the cells by adding it as either
an oleate/BSA complex (7:1, mol/mol) or a palmitate/BSA complex (5:1,
mol/mol) alone or in combination with 5 mM glucose. The
results show that oleate, like palmitate (2), potentiated
glucose-stimulated secretion (P < 0.001) and had
little effect in the absence of glucose (Fig 5). The effect of PKC down-regulation on
this potentiation was of interest because down-regulation removes all
cPKC activity and a majority of nPKC activity found in this clonal
ß-cell (Fig. 1). PKC down-regulation did not alter FFA potentiation
of glucose-stimulated secretion (P < 0.01
vs. control), while the effect of glucose itself was
enhanced (P < 0.01 vs. control) (Fig. 5).
The loss of the acute action of PMA or 1,2-dioctanoylglycerol to
potentiate glucose-stimulated secretion served as a positive control
for PKC down- regulation in these cells (data not shown).
To determine which PKC isoforms were expressed in the clonal ß-cells
and which remained following PMA-induced down-regulation, Western
blotting was performed (Fig. 6). The cPKC
isoforms, and ßII, the nPKC isoforms, and 
, as well as the
aPKC isoforms, and , were expressed in this clonal ß-cell
(Fig. 6). In addition to these isoforms PKC-µ, a member of the PKD
class of kinases, was also present (data not shown). cPKC-ßI and ,
the nPKC-
, and the aPKC-
were probed for and not found. As
expected PMA down-regulation removed the conventional isoforms and
ßII and the novel isoform while not affecting the atypical
isoforms and . Unexpectedly, PKC- was also not down-regulated
with its mass possibly increased by exposure to PMA. These effects of
PMA correlate with the complete loss of Ca2+
stimulated activity and the partial loss of Ca2+
independent activity as shown in Fig. 1.
The distribution between soluble and particulate fractions of isoforms
illustrated in Fig. 6, differed among isoforms. Fractions were prepared
in buffer containing the Ca2+ chelator EGTA. cPKC
isoforms were restricted to the cytosol in the absence of
Ca2+ as was the nPKC isoform . The other nPKC
isoform expressed, PKC-, appeared associated with the cytosol, the
detergent-soluble membranes and the cytoskeleton fractions (insoluble
protein). The aPKC isoforms also demonstrated differential distribution
with little or no PKC- associated with the cytosol fraction, while
PKC- was recovered in all fractions. These differences in isoform
distribution in the absence of Ca2+ could be
explained by differences in the mechanism targeting these isoforms to
intracellular membranes (16).
To determine whether translocation of the aPKC isoform might play a
role in glucose and FFA stimulated insulin secretion, the intracellular
distribution of this aPKC was examined following acute stimulation by
glucose or glucose and oleate. In Fig. 7, a representative Western blot illustrates the enrichment of a total
membrane pellet with PKC- seen 3 min following cell stimulation. The
corresponding relative pellet densities, normalized to the basal
pellet, are shown below. This experiment was repeated five times with
transient increases in PKC- mass occurring in the membrane fraction
in all experiments between 1 and 5 min following stimulation. Both
glucose and glucose plus oleate caused translocation with the latter
condition being more effective in four of the five experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most dramatic action of the LC-CoA esters was their ability to
stimulate aPKC activity, presumably PKC- or , in the presence of
micelles containing PS. This activation was not dependent upon
Ca2+ or altered by PMA-induced down-regulation.
These results are consistent with the possibility that aPKCs are
regulated in vivo by LC-CoA esters and that CoA derivatives
could be the physiologically relevant activator of this PKC class.
While the cytosolic free concentration of LC-CoA esters is not known
for any tissue the intracellular concentration has been estimated to be
as high as 90 µM in clonal ß-cells with total
cell concentrations in other tissues reported to range from 5–160
µM (24, 25). Assuming that the cytosol water
space in the ß-cell is similar to the hepatocyte, a total cytosolic
concentration of 76 µM would be predicted (24).
The question of free concentration of LC-CoA would then depend on both
the affinity and number of binding sites, such as specific binding
proteins and membranes, present in cells and in the ß-cell has been
estimated to be 0.5 µM (24). In the assay used
here additional high affinity binding sites were provided by the
phospholipid-containing micelles used (25). Because PKC is activated on
the surface of micelles or membranes raises the question of whether
bound or free LC-CoA molecules are important in this mechanism. This
total cytosolic value is within the range used in the assays reported
here.
In contrast to their effects on aPKC activity, our data predict
that nPKC activity would be inhibited by a rise in cytosolic LC-CoA.
Because the membrane content of DAG also increases as a consequence of
FFA metabolism, it is difficult to predict the net effect on nPKC
activity. Previous studies have demonstrated both stimulatory and
inhibitory effects of LC-CoA esters on PKC activity. In rat brain,
total PKC activity (PS, DAG and Ca2+) was
stimulated by palmitoyl-CoA, oleoyl-CoA and myristoyl-CoA (26, 27).
Interestingly, palmitoyl-CoA was without effect in the presence of PS
and Ca2+ alone, possibly because brain extract
does not contain aPKC isoforms or their activation by palmitoyl-CoA was
inhibited by Ca2+. However, brain PKC activity
was stimulated approximately 4-fold rather than the doubling of the
activity seen here. The difference in stimulation may be due to the
level of PS used in the two studies. In the present study, the mol %
of PS was 15.6%, whereas it ranged from zero up to 9 mol % in the
aforementioned brain study. At the highest level of PS in the brain
study and in the presence of 100 µM palmitoyl-CoA, cPKC
activity was stimulated to a similar extent as in Fig. 2. The
inhibition of nPKC activity reported here is in accord with our
previous demonstration of inhibition of partially purified nPKC by
oleoyl-CoA and myristoyl-CoA from neutrophils (19).
The mechanism by which LC-CoA modulates the activity of PKC is unknown. The lack of effectiveness seen with the short chain CoA esters, succinyl-CoA and malonyl-CoA, suggests that only LC-CoA are effective. Three possible explanations are that: 1) there is a LC-CoA binding or acylation site(s) on the enzyme; 2) the acyl-CoA is needed to allow PKC to insert into the membrane; or 3) that the activity of the enzyme is modulated secondary to changes in physical characteristics of the lipid bilayer. It is interesting that at 10 µM both myristoyl- and oleoyl-CoA inhibit nPKC, whereas palmitoyl-CoA is without effect. Perhaps this is due to the solubility of the different LC-CoA and their equilibrium between the free monomer and micelles. The data are consistent with the concentration of LC-CoA monomer being important. Alternatively, palmitoyl-CoA could affect different nPKC isoforms in an opposite manner with the net effect being zero.
The action of the FFA itself may also be important in this setting as there are reports of FFA translocating PKC within a cell or the in vitro activation of PKC (28, 29, 30, 31). The small stimulation of nPKC activity seen with oleic acid would be consistent with the reported activation of soluble PKC by FFA (28) but appears distinct from the inhibition observed here. Background kinase activity was stimulated by oleic acid, but this activation was unaffected by the addition of PS. This results suggests that the kinase involved is not PKC and may be another lipid activated kinase. Whatever its identity, this kinase activity would be increased as extracellular FFA partitioned into cell membranes.
Our secretion data demonstrate that exogenous oleate and
palmitate potentiated glucose-induced insulin secretion and that
stimulated secretion and its potentiation were preserved after
PMA-induced down-regulation of the ß-cells. These data have several
implications regarding the possible role PKC isoforms in secretion.
First, glucose-induced secretion and its potentiation could not be due
to activation of cPKC isoforms as PMA down-regulation caused the
complete loss of Ca2+-dependent activity and
isoform mass as shown in Figs. 1 and 6, respectively. Second, while
palmitate and oleate had similar effects on secretion, their acyl-CoA
derivatives had distinctly different effects on nPKC activity.
Therefore, nPKC isoforms are unlikely mediators of the potentiation
observed. Third, the most prominent effect of oleoyl-CoA or
palmitoyl-CoA was to stimulate aPKC activity that was unaltered by PMA
down-regulation (Fig. 6). It is not clear why oleate and palmitate act
similarly on secretion while oleate is more potent on aPKC than
palmitate. Physical differences in the addition of a FFA/BSA complex to
intact cells vs. the addition of a water soluble LC-CoA
derivative to the PKC assay may account for the quantitative
differences seen in their response (Fig. 5 vs. Fig. 3).
Previous work indicates that both glucose and FFA raise cytosolic
LC-CoA levels in the ß-cell, whereas the data presented here predicts
that aPKC isoforms would be activated by this increase. One proxy for
the activation of PKC in a cellular process is its translocation to a
membrane compartment following cell stimulation. The enrichment of a
total membrane fraction by PKC- (Fig. 7) following stimulation by
either glucose or glucose plus oleate is consistent with this model and
with the PKC- translocation that occurs in carbachol-stimulated
insulin secretion (32). Even though the mechanism of translocation my
differ between nutrient and acetylcholine-induced secretion, these
results suggest a role for PKC- in secretion. It is potentially
significant that the observed translocation peaked only transiently at
various times between 1 and 5 min following cell stimulation. Perhaps
this variation in timing reflects the reported rapid and transient
nature of PKC movement following FFA exposure (33) or the oscillatory
nature of glucose-stimulated insulin release (34). The physiological
significance of the interaction between extracellular FFA and glucose
is emphasized by the requirement of the perfused pancreas from starved
rats for extracellular FFA to remain glucose responsive (35).
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Acknowledgments |
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Footnotes |
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Received August 30, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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overexpressed in COS cells: comparison with other protein kinase C
isotypes and other acidic lipids. Biochem J 304:1001–1008
mediates carbachol-stimulated insulin secretion in
RINm5F cells. Diabetes 47:905–912[Abstract]
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K. Fujiwara, F. Maekawa, and T. Yada Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet {beta}-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release Am J Physiol Endocrinol Metab, October 1, 2005; 289(4): E670 - E677. [Abstract] [Full Text] [PDF]
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